1. Field of the Invention
This invention provides methods for toxicological screening of pharmaceuticals and other chemical compounds. The invention specifically provides reagents that are human embryonic stem cells (hESC) or hESC-derived lineage-specific cells, such as neural stem cells, neural precursor cells and neural cells, as well as methods for using these cells to detect developmental toxicity or teratogenic effects of pharmaceutical compounds and other chemicals. More particularly, the invention provides an in vitro means for analyzing toxicity of compounds predictive of their toxicity during human development. Candidate predictive biomarkers for toxic or teratogenic effects are also identified and provided herein.
2. Background of Invention
Birth defects are a major cause of infant morbidity in the United States, affecting 1 in every 33 infants born (Brent & Beckman, 1990, Bull NY Acad Med 66: 123-63; Rosano et al., 2000, J Epidemiology Community Health 54:660-66), or approximately 125,000 newborns per year. It is understood that developmental toxicity can cause birth defects, and can generate embryonic lethality, intrauterine growth restriction (IUGR), dysmorphogenesis (such as skeletal malformations), and functional toxicity, which can lead to cognitive disorders such as autism. There is an increasing concern about the role that chemical exposure can play in the onset of these disorders. Indeed, it is estimated that 5% to 10% of all birth defects are caused by in utero exposure to known teratogenic agents (Beckman & Brent, 1984, Annu Rev Pharmacol 24: 483-500).
Concern exists that chemical exposure may be playing a significant and preventable role in producing birth defects (Claudio et al., 2001, Environm Health Perspect 109: A254-A261). This concern has been difficult to evaluate, however, since the art has lacked a robust and efficient model for testing developmental toxicity for the more than 80,000 chemicals in the market, plus the new 2,000 compounds introduced annually (General Accounting Office (GAO), 1994, Toxic Substances Control Act: Preliminary Observations on Legislative Changes to Make TSCA More Effective, Testimony, Jul. 13, 1994, GAO/T-RCED-94-263). Fewer than 5% of these compounds have been tested for reproductive outcomes and even fewer for developmental toxicity (Environmental Protective Agency (EPA), 1998, Chemical Hazard Data Availability Study, Office of Pollution Prevention and Toxins). Although some attempts have been made to use animal model systems to assess toxicity (Piersma, 2004, Toxicology Letters 149:147-53), inherent differences in the sensitivity of humans in utero have limited the predictive usefulness of such models. Development of a human-based cell model system would have an enormous impact in drug development and risk assessment of chemicals.
Toxicity, particularly developmental toxicity, is also a major obstacle in the progression of compounds through the drug development process. Currently, toxicity testing is conducted on animal models as a means to predict adverse effects of compound exposure, particularly on development and organogenesis in human embryos and fetuses. The most prevalent models that contribute to FDA approval of investigational new drugs are whole animal studies in rabbits and rats (Piersma, 2004, Toxicology Letters 149: 147-53). In vivo studies rely on administration of compounds to pregnant animals at different stages of pregnancy and embryonic/fetal development (first week of gestation, organogenesis stage and full gestation length). However, these in vivo animal models are limited by a lack of robustness between animal and human responses to chemical compounds during development. Species differences are often manifested in trends such as dose sensitivity and pharmacokinetic processing of compounds. At present, animal models are only 50% efficient in predicting human developmental response to compounds (Greaves et al., 2004, Nat Rev Drug Discov 3:226-36). Thus, human-directed predictive in vitro models present an opportunity to reduce the costs of new drug development and enable safer drugs.
In vitro models have been employed in the drug industry for over 20 years (Huuskonen, 2005, Toxicology & Applied Pharm 207:S495-S500). Many of the current in vitro assays involve differentiation models using primary cell cultures or immortalized cells lines (Huuskonen, 2005, Toxicology & Applied Pharm 207:S495-S500). Unfortunately, these models differ significantly from their in vivo counterparts in their ability to accurately assess development toxicity. In particular, the ECVAM initiative (European Center for Validation of Alternative Methods) has used mouse embryonic stem cells as a screening system for predictive developmental toxicology. The embryonic stem cell test (EST) has shown very promising results, with a 78% statistically significant correlation to in vivo studies, and the test was able to differentiate strong teratogens from moderate/weak or non-embryotoxic compounds (Spielmann et al., 1997, In Vitro Toxicology 10:119-27). This model is limited in part because toxicological endpoints are defined only for compounds that impair cardiac differentiation. This model also fails to account for interspecies developmental differences between mice and humans, and so does not fully address the need in the art for human-specific model systems.
Thus there remains a need in this art for a human-specific in vitro method for reliably determining developmental toxicity in pharmaceutical agents and other chemical compounds. There also is a need in the art to better understand human development and its perturbation by toxins and other developmental disrupting agents, to assist clinical management of acquired congenital disorders and the many diseases that share these biochemical pathways, such as cancer.
The present invention provides for the assessment of a plurality of small molecules, preferably secreted or excreted by hES cells or hESC-derived lineage-specific cells, such as neural stem cells, neural precursor cells and neural cells, and is determined and correlated with health and disease or insult state. Similar analyses have been applied to other biological systems in the art (Want et al., 2005 Chem Bio Chem 6: 1941-51), providing biomarkers of disease or toxic responses that can be detected in biological fluids (Sabatine et al., 2005 Circulation 112:3868-875).